Lateral-flow immunoassay tests have been in wide use since their introduction in the mid-1980's [Rosen]. The device format has proven to be very popular due to its flexibility, low cost, ease and speed of use, and rapid development cycle for new assays. Lateral flow devices are usually comprised of a plastic housing and a porous test strip. The housing protects and binds the components of the test strip together, provides a controlled means for sample application to the test strip, and provides indicator markings and directions for use. The sample strip is the heart of the device, usually comprised of four separate, but overlapping strips, allowing the sample fluid to flow by capillarity from one strip, or zone, to the next [O'Farrell]. The first zone is the sample pad, which draws sample fluid into the device and may perform treatments (filtration, buffering, etc.) necessary for the assay to function. The second zone is the conjugation pad, containing certain dried components for the assay. In the case of sandwich-type immunoassays, these components will usually be the detection part of the sandwich—monoclonal antibodies specific for the analyte that are conjugated to a detection agent, such as an enzyme, or colored latex microbeads. These components are solubilized by the sample fluid, and available for reaction. The next zone is the reaction matrix, containing components of the assay necessary for reading a result, usually immobilized in well-defined detection zones for easy readout as visible indicator lines. In the case of sandwich-type immunoassays, these components would usually be polyclonal antibodies specific to the analyte. The last zone is the wick, which soaks up excess sample fluid and keeps fluid flowing through the rest of the sample strip until the reaction reaches completion.
These assays can also use enzyme-mediated reporter formats. One example of this can be demonstrated by an assay to measure glucose in a sample fluid. Enzymes glucose oxidase and horseradish peroxidase can be bound to a region of a test strip. The sample can be mixed in a known ratio with a substrate such as 3,3′-Diaminobenzidine (DAB) and the sample applied to the test strip. As the sample migrates toward and through the region with the immobilized enzymes, the glucose contained within the sample will react with the glucose oxidase producing hydrogen peroxide and d-Gluconic Acid. In turn, the hydrogen peroxide and the DAB will react with the horseradish peroxidase to produce an insoluble brown-black precipitate which will be visible as an indicator line.
The above assays can also be run in competitive formats, useful when there may be high concentrations of analyte in the sample fluid, or cases when sandwich assays are not feasible, such as small molecule analytes. In one example of this strategy, polyclonal antibodies specific to the analyte would be immobilized at the indicator lines. The conjugate pad would include latex beads coated with analyte. In the absence of analyte, the beads would bind at the indicator lines as the sample flows through to produce a readable signal. In the presence of high concentrations of analyte in the sample fluid, the analyte would compete for the immobilized antibodies in the detection zone with the analyte bound to the beads, producing a much reduced signal.
The assays described above depend on an accumulation of reporter molecule at a specific point on the test strip to form indicator lines. The flow rate of the sample fluid through the detection zone in effect determines the incubation time, and thus determines the minimum reaction constants that the antibodies must have [Brown]. Too high of an analyte concentration in the sample can actually lead to a drop-off in signal by as much as two orders of magnitude, as the sample saturates both the capture and indicator antibodies without forming sandwiches, a phenomenon known as “sensor poisoning” [Qian]. Optimal conditions depend on many factors, including flow rate, and therefore much design time is spent trying to minimize the effect of varying flow rate.
In contrast, the devices described herein rely on changes in flow rate and/or measurement of a fluid front as an analytical tool.